Use of Timosaponin BII in the Preparation Of A Medicament or Product for the Prevention and Treatment of Stroke

ABSTRACT

The invention disclosed the use of timosaponin BII in the preparation of a medicament or product for the prevention and treatment of stroke. The experiments prove that timosaponin BII can improve the neurological symptoms of cerebral ischemic rat, reduce infarct size, relieve brain water edema, improve hemorheology, reduce inflammatory injury of cerebral ischemia.

TECHNICAL FIELD

The present invention relates to use of timosaponin BII, in particularthe use of timosaponin BII in the preparation of a medicament or productfor the prevention and treatment of stroke (apoplexy).

BACKGROUND ART

Stroke (apoplexy) involves sudden loss of neural function due todisturbance in cerebral perfusion owing to intracranial and extracranialangiemphraxis or angiorrhexis, which seriously harms the health of theaged and middle-aged as a main cause of disability and death. About onethird of patients attacked by the disease will develop to death; evensurvivors will lose their work ability or self-care ability due tosequelae such as hemiplegia and aphasia. At present, there are twotreatments available for stroke: One is to relieve deficiency of oxygenand glucose in arteries via increasing blood flow; the other is toprotect neuron through blocking neuron death caused by cerebral ischemiaand excitotoxicity. Clinically used neuron protective agents includecalcium channel blockers, glutamate receptor antagonists and NMDAantagonists etc. However, in view of the high morbidity, mortality anddisability rates, there are few drugs developed for prevention andtreatment of stroke, which is far from enough to meet clinical need.

Rhizome Anemarrhenae as an herbal medicine, is rhizome of Anernarrhenaasphodeloides Bge, anemarrhena of family Liliaceae, which is mainlyproduced in Hebei, Inner Mongolia, Shanxi and northeast China. Intraditional Chinese medicine, it is used as a bitter cold heat-clearingdrug with effects of relieving exogenous febricity, hyperpyrexia andpolydipsia, lung-heat and dry cough, osteopyrexia and fevers calorintemus and diabetes and dryness of the intestine and constipation.Essential component of anemarrhenae is steroidal saponins. To date, 32kinds of steroidal saponins and sapogenin isolated from anemarrhenaehave been reported as well as other components such as chromocor,oligosaccharide, polysaccharides, fatty acid, etc. Kawasaki et al. firstisolated the timosaponin BII in 1963, but did not elucidate its chemicalstructure. Seiji Nagumo et al. elucidated the chemical structure oftimosaponin BII first in 1991 (Seiji NAGUMO et al, J. Pharm. (Japanese),1991: 111(1); 306-310). From then on, extraction and activitydetermination of Timosaponin BII were reported by Noboru Nakashima(NOBORU NAKASHIMA et al, Journal of Natural Products, 1993; 56(3):345-350), Ma Bai ping (Ma B P et al., Yao Xue Xue Bao, 1996: 31(4):271-277), Masayasu Kimula (Masayasu KIMURA et al, Biol. Pharm. Bull,1996; 19(7); 926-931), Jianying Zhang (Jianying ZHANG et al, ClinicaChimica Acta, 1999; 289: 79-88) successively.

Timosaponin BII, also called Prototimosaponin AIII, is the essentialcomponent of anemarrhenae. Its chemical name is(25S)-26-O-β-D-glucopyranosyl-22-hydroxy-5β-furostane-3β,26-diol-3-O-β-D-glucopyranosyl(12)-β-D-galactopyranoside,with structural formula shown as follow:

The pharmacological activities of Timosaponin BII that have beenreported mainly include:

1. Hypoglycemic activity. Timosaponin BII can lower blood sugar level instreptozocin-induced diabetic mouse without promoting absorption ofglucose and release of insulin. The mechanism of reducing blood glucoseis supposed to be the inhibition of liver sugar decomposition (NOBORUNAKASHIMA et al, Journal of Natural Products, 1993; 56(3): 345-350)2. Inhibition of platelet aggregation. Timosaponin BI possesses activityof obviously inhibiting platelet aggregation and prolonging clottingtime (Jianying ZHANG et al, Clinica Chimica Acta, 1999; 289:79-88).3. Clearance of free radicals. Observation via paramagnetic method showsthat timosaponin BII can clear 57% free radicals generated from Fentonreaction system (Ma B P, et al. Yao Xue Xue Bao, 1996; 31(4): 271-277).4. Anti-dementia activity. Ma Bai ping et al. reported that timosaponinBII have preventive and therapeutic effect against senile dementia(Chinese Patent Application Publication No. CN1212966, Application No.97119680.X).

Chen Wan sheng et al. reported the use of total Timosaponins inpreparation of medicaments for prevention and treatment of stroke(Chinese Patent Application Publication No. CN1451384A Application No.03116824.8). The total timosaponins disclosed therein are characterizedby a sum of contents of timosaponins BII, E, B, AIII of ≧50%.

CONTENTS OF THE INVENTION

After years of intensive research, the present inventors first found andconfirmed the activity of timosaponin BII alone in the prevention andtreatment of stoke. Said compound can significantly ameliorateneurological symptoms in ischemic rats, reduce size of cerebralinfarction and relieve the extent of cerebral edema; and significantimprove the hemorheology in the model animals and reduce inflammatoryinjury caused by cerebral ischemia.

Therefore, the present invention relates to the use of timosaponin BIIin the preparation of a medicament or product for the prevention ortreatment of stroke.

The present invention also relates to a pharmaceutical composition orproduct for the prevention or treatment of stroke, comprisingtimosaponin BII as the active ingredient and a pharmaceuticallyacceptable carrier or excipient.

According to the present invention, timosaponin BII is used in asubstantially pure form, e.g. the purity of timosaponin SU being ≧90%.

According to the present invention, the pharmaceutical composition orproduct comprising timosaponin BII can be administered through variousroutes, and formulated into various dosage forms, e.g. oral dosageforms, such as a tablet, capsule, solution, suspension; parenteraldosage forms, such as an injection, ointment, patch, etc. According tothe present invention, continuous administration of timosaponin BIT (20mg/kg and 40 mg/kg) for 7 days could significantly lower theneurological symptom score in rats having middle cerebral artery (MCA)thrombosis, and significantly reduce the cerebral infarct size in themodel rats; administration at 40 mg/kg could significantly reduce thewater content of brain when compared with the model group (p<0.05,p<0.01).

According to the present invention, subcutaneous injection of adrenalineplus ice-water immersion method could lead to an acute blood stasis ratmodel, and administration of timosaponin BII (10 mg/kg, 20 mg/kg, 40mg/kg) could significantly reduce the whole blood and plasma viscositiesunder high, middle and low shear rates, as compared with the model group(p<0.05, p<0.01). Timosaponin BII at 40 mg/kg could significantlyimprove the deformation ability of erythrocytes, and reduce theerythrocyte agglutination index, as compared with the model group(p<0.05, p<0.01).

The embolic thread method was employed in the preparation ofcerebral-ischemia-reperfusion rat model, and ELISA was used to determinethe levels of IL-1β, TNF-α, IL-10 and TGF-β in each group. The resultsshowed that timosaponin BII had significant protective effect againstthe inflammatory responses in ischemia-reperfusion model rats.

To sum up, timosaponin BII has utility in the prevention and treatmentof stroke.

MODES OF CARRYING OUT THE INVENTION

The following examples are intended to further illustrate the invention,but not meant to be limiting in any way.

Example 1 Effect of Timosaponin BII on Ischemic Cerebral Injury inFeCl₃-Induced Middle Cerebral Artery Thrombosis (MCAT) Rats I. Method 1.Effect of Timosaponin BII on Neurological Symptoms and Cerebral InfarctSize in Middle Cerebral Artery Thrombosis Rats 1.1. Grouping and Dosing

The experimental animals were randomly divided into 6 groups, i.e. MCATmodel group, sham-operation group, hydergine (0.6 mg/kg) group,timosaponin BII 10 mg/kg group, 20 mg/kg group and 40 mg/kg group.Continuous administration of the drugs by gavage was performed for 7days with daily volume of 5 ml/kg. The operation was carried out onehour after drug administration on the seventh day. The MCAT model andsham-operation groups were given the same volume of a 0.5% CMC solution.

1.2. Preparation of MCA Thromboembolia-Induced Ischemic Cerebral InjuryModel in Rats

Tamura and Liu's methods were used with modifications to prepare themodel. Rats were anesthetized by intraperitoneal injection of 10%chloral hydrate (0.35 g/kg); and fixed with right arm reclining. An arcincision of about 1.5 cm in length was made at the midpoint between thelink of paropia and external auditory canal and temporalis was removedto expose the temporal bone. An opening in the bone of about 2.5 mm indiameter was made by a bur drill at the junction of malar bone and quamatemporalis 1 mm cephalically under stereoscopic microscope, and residueswere cleaned up to expose the middle cerebral artery (located betweenthe olfactory tract and inferior cerebral vein). The surrounding-tissueswere protected with a piece of plastic film. A small piece ofquantitative filter paper, which had absorbed 10 μL of a 50% ferricchloride solution, was applied on this segment of middle cerebralartery. The filter paper was removed after 30 min, and local tissueswere rinsed with normal saline solution, followed by suture layer bylayer. Then the experimental rats were put back to rearing cage. Thetemperature was controlled at 24° C. For the sham-operation group, theoperation procedures were the same as the model group except for theapplication of the ferric chloride solution.

1.3. Assessment of Neurological Symptom Score

The experimental animals were tested for behaviors 6 h and 24 h afteroperation according to the Sederson's method with modifications.Criteria: {circle around (1)} The tail of the rat was lifted to observethe flexibility of the forelimb, with both forelimbs symmetricallystretching forward scored as 0, and occurrence of any or all of shoulderinflexion, elbow inflexion, shoulder intorsion in the contralateralfore-limb opposite to the operation side scored as 1. {circle around(2)} The animal was placed on a plane, and its shoulders were pushed tothe opposite direction to check resistance, Equivalency and strongresistance in both sides was scored as 0, and reduction in resistance inthe contralateral side was scored as 1. {circle around (3)} Bothforelimbs of the rat were placed on wire gauze to observe the musculartension. Equivalency and strong tension in both sides was scored as 0,and reduction in muscular tension in the contralateral forelimb wasscored as 1. {circle around (4)} The tail of the rat was pulled up, andthe animal exhibiting incessant rotations in the direction opposite tothe operation side was scored as 1. According to the criteria above, thefull score is 4, and the higher the score is, the more serious thebehavior disorder of the animal is.

1.4. Determination of Cerebral Infarct Size

After behavior assessment, the animals were decapitated to remove thebrains. The olfactory bulb, cerebellum and lower brain stem wereremoved, and the remaining part was cut into 5 coronal sections below 4°C. The sections were immediately placed into a TTC staining solution(containing 1.5 m 4% TTC, 0.1 ml 1M K₂HPO₄ and water (balance) per 5 mlstaining solution), followed by incubation in dark at 37° C. for 30 min.Then, the sections were transferred into a solution of 10% formaldehydeand incubated in dark for 24 h. After staining, the non-ischemic areaswere rose red, and the infracted areas were white. The white tissueswere carefully collected and weighed. The extent of cerebral infarctionwas expressed as a percentage (%) of infracted tissue to the weight oftotal brain and also to the affected side of brain.

2. Effect of Timosaponin BII on Water Content in Brain Tissues in MiddleCerebral Artery Thrombosis (MCAT) Rats 2.1. Grouping, Dosing andModelling Methods (See 1 Above.) 2.2. Determination of Water Content inBrain Tissues

The rats were decapitated 24 h after operation, and the brain wereremoved and separated into left and right hemispheres. Filter paper wasused to blot surface and the wet weight of each hemisphere was weighed,respectively. Then, drying of the brain tissues at 105° C. was performedfor 48 h followed by accurate weighing of the dry weight. The watercontent of the brain was calculated according to the following formula:

Water content of brain (wet weight−dry weight)/wet weight×100%

3. Statistical Analysis

The experimental data were expressed as x±s. The results were analysedusing SPSS software and t-test was used to perform comparison amonggroups.

II. Results 1. Effect of Timosaponin BII on Neurological Symptoms andCerebral Infarct Size of Middle Cerebral Artery Thrombosis Rats

The results are shown in Table 4-1 below.

TABLE 4-1 Effect of timosaponin BII on neurological symptoms andcerebral infarct size of middle cerebral artery thrombosis rats ( x ± s)cerebral infarct size percentage percentage to neurological Dosage tototal brain affected side symptomatic score Group mg/kg n (%) (%) 6 h 24h Sham-operation — 12   0 ± 0   0 ± 0 0.08 ± 0.29 0.17 ± 0.39 MCAT model— 12 4.58 ± 1.59^(ΔΔ) 8.76 ± 2.98^(ΔΔ) 2.73 ± 1.01^(ΔΔ) 2.55 ± 0.52^(ΔΔ)Hydergine 0.6 11 1.98 ± 0.72** 3.75 ± 1.38** 1.91 ± 0.83* 1.55 ± 0.69**Timosaponin BII 10 12 3.27 ± 1.74 6.35 ± 3.39 2.08 ± 0.67 2.08 ± 0.79Timosaponin BII 20 11 2.92 ± 1.20* 5.37 ± 2.20* 1.82 ± 0.75* 2.00 ±0.45* Timosaponin BII 40 10 1.99 ± 1.20** 3.88 ± 2.38** 1.50 ± 0.71**1.90 ± 0.74* Notes: ^(ΔΔ)P < 0.01 vs. sham-operation group; *P < 0.05,**P < 0.01 vs. the model group.

The results indicate that as compared with the sham-operation group,animals of MCAT model group and each administration groups exhibitedinfarction focus and hemiplegia like symptoms to various extents.Timosaponin BII at 20 mg/kg and 40 mg/kg significantly reduced thecerebral infarct size and ameliorate neurological symptoms in modelanimals (P<0-05, P<0.01).

2. Effect of Timosaponin BII on Water Content in Brain Tissues in MiddleCerebral Artery Thrombosis (MCAT) Rats

The results were shown in Table 4-2 below.

TABLE 4-2 Effect of timosaponin BII on water content in brain tissues inmiddle cerebral artery thrombosis (MCAT) rats ( x ± s) Water content ofWater content of Dosage unaffected affected Group (mg/kg) n hemisphere(%) hemisphere (%) Sham-operation — 12 79.60 ± 0.49 79.59 ± 0.48 MCATmodel — 12 79.54 ± 0.80 81.12 ± 0.90^(ΔΔ) Nimodipine 12 12 79.44 ± 0.4180.37 ± 0.73* Timosaponin BII 10 12 79.51 ± 0.57 80.52 ± 0.69Timosaponin BII 20 12 79.41 ± 0.38 80.46 ± 0.79 Timosaponin BII 40 1279.47 ± 0.44 80.15 ± 0.99* Notes: ^(ΔΔ)P < 0.01 vs. the sham-operationgroup; *P < 0.05 vs. the model group.

As indicated in the table above, timosaponin BII at 40 mg/kdsignificantly lowered the water content in the affected hemisphere andameliorated cerebral edema in model rats (P<0.05).

Discussion and Summary

Middle cerebral artery thrombosis model is a common animal model offocal cerebral ischemia, which objectively simulates clinicalcircumstance of cerebral infarction in Middle cerebral artery, withadvantages of easy control for local conditions and replication,similarity to the course of clinical cerebral apoplexy and fixedlocation of thrombus. As the results show, 6 h or 24 h after operation,hemiplegia like symptoms, increase of water content in cerebralthrombosis side and marked cerebral infarction (by TTC staining) wereobserved. As compared with the sham-operation group timosaponin BII at20 mg/kg and 40 mg/kg group significantly reduced cerebral infarct sizeand ameliorate neurological symptoms (P<0.05, P<0.01). Additionally, ascompared with the model group, extent of cerebral edema in timosaponinBII 40 mg/kg group was significantly reduced (P<0.05). The resultssuggest that the drug has protective effect against ischemic cerebralinjury.

Example 2 Effect of timosaponin BII on Hemorheology in Acute BloodStagnation Model Rats I. Method 1. Grouping and Dosing

The experimental animals were randomly divided into 6 groups based onweight, i.e. model group, normal control group, nimodipine (12 mg/kg)group, timosaponin BII 10 mg/kg group, 20 mg/kg group and 40 mg/kggroup. Continuous administration of the drugs by gavage was performedfor 5 days with daily volume of 5 ml/kg. The normal control group andthe model group were given the same volume of a 0.5% CMC solution.

2. Modelling Method

MAO Teng-min's methods were modified to prepare the model. Rats weresubcutaneously injected with 0.8 mg/kg adrenaline twice at 1 h and 5 hafter administration on the fifth day, and were placed into ice-water at4° C. for 5 min at 2 h after the first injection of adrenaline.

3. Assessment of Whole Blood and Plasma Viscosities

Rats were anaesthetized by intraperitoneal injection of 10% chloralhydrate (0.35 g/kg) 1 h after the last administration. Blood was drawnfrom carotid artery and treated with 1% heparin. 0.8 ml ofanticoagulated blood was tested in a blood viscometer and whole bloodviscosity was represented by blood viscosity tested at high (200 S⁻¹),middle (30 S⁻¹), low (5 S⁻¹) and low (1 S⁻¹) shear rates. In addition,anticoagulated blood was centrifuged at 300 rpm for 8 min to yieldplasma as the supernatant, and 0.8 ml of plasma was test for plasmaviscosity at 100 S⁻¹ in a blood viscometer.

4. Determination of Erythrocyte Aggregation and Deformability

To 40 μl of heparinized blood, 1 ml of deforming solution was added andmixed thoroughly. A sample of 0.8 ml was tested in an erythrocyteaggregation/deformability tester, and erythrocyte deformability wasexpressed as maximal erythrocyte deformation index and area under curve(SSS). Another 0.8 ml of anticoagulated blood was tested in anerythrocyte aggregation/deformability tester, and erythrocyteaggregation was expressed as maximal erythrocyte aggregation index(MAXD) and area under curve (SSS).

5. Statistical Analysis

The experimental data were expressed as x±s. The results were analysedusing SPSS software and t-test was used to perform comparison amonggroups.

II. Results 1. Effect of Timosaponin BII on Whole Blood and PlasmaViscosities in Acute Blood Stagnation Model Rats

The results are shown in Table 10-1 below.

TABLE 10-1 Effect of timosaponin BII on whole blood and plasmaviscosities in acute blood stagnation model rats ( x ± s) Plasma DosageWhole blood viscosity viscosity Group mg/kg n 200 S⁻¹ 30 S⁻¹ 5 S⁻¹ 1 S⁻¹(100 S⁻¹) Blank control — 13 3.81 ± 0.42 5.42 ± 0.60 10.34 ± 1.41 25.74± 4.64 1.33 ± 0.11 Model — 14 5.05 ± 0.74^(ΔΔ) 7.56 ± 1.18^(ΔΔ) 15.52 ±3.28^(ΔΔ) 41.48 ± 11.64^(ΔΔ) 1.53 ± 0.11^(ΔΔ) Nimodipine 12 12 4.51 ±0.55* 6.47 ± 0.92* 12.34 ± 2.58** 30.45 ± 8.74* 1.41 ± 0.14* TimosaponinBII 10 10 4.45 ± 0.71 6.10 ± 0.85** 11.02 ± 1.38** 27.03 ± 4.64** 1.40 ±0.10** Timosaponin BII 20 13 4.49 ± 0.70 6.55 ± 0.93* 12.91 ± 1.82*33.13 ± 5.70* 1.36 ± 0.10** Timosaponin BII 40 12 4.42 ± 0.38* 6.45 ±0.60** 12.46 ± 1.33** 31.21 ± 4.29** 1.38 ± 0.12** Notes: ^(ΔΔ)P < 0.01vs. the blank control group; *P < 0.05, **P < 0.01 vs. the model group.

The results indicate that as compared with the normal control group,whole blood and plasma viscosities significantly increased in bloodstagnation model rats at 24 h after modelling (P<0.01). Timosaponin BIIat 10 mg/kg, 20 mg/kg and 40 mg/kg significantly lowered whole blood andplasma viscosities at high, middle and low shear rates in model rats(P<0.05, P<0.01).

2. Effect of Timosaponin BII on Erythrocyte Aggregation andDeformability in Acute Blood Stagnation Model Rats

The results are shown in Table 10-2 below.

TABLE 10-2 Effect of timosaponin BII on erythrocyte aggregation anddeformability in acute blood stagnation model rats ( x ± s) DosageErythrocyte aggregation Erythrocyte deformability Group (mg/kg) n MAXDSS MAXDI SSS Blank Control — 13 0.52 ± 0.13  94.97 ± 25.97 0.71 ± 0.03365.02 ± 20.48 Model — 14 0.89 ± 0.11^(ΔΔ) 176.52 ± 19.74^(ΔΔ) 0.69 ±0.02^(Δ) 349.22 ± 10.59^(Δ) Nimodipine 12 12 0.80 ± 0.09* 160.38 ±16.50* 0.70 ± 0.03 353.92 ± 15.83 Timosaponin 10 10 0.94 ± 0.14 186.67 ±28.85 0.72 ± 0.05 363.88 ± 19.58 BII Timosaponin 20 13 0.81 ± 0.26163.04 ± 50.23 0.71 ± 0.05 363.97 ± 27.87 BII Timosaponin 40 12 0.76 ±0.14* 152.78 ± 28.11* 0.73 ± 0.03** 374.26 ± 15.82** BII Notes: ^(Δ)P <0.05, ^(ΔΔ)P < 0.01 vs. the blank control group; *P < 0.05, **P < 0.01vs. the model group.

The results indicate that as compared with the blank control group,blood stagnation model rats showed significant increased erythrocyteaggregation index and decreased erythrocyte deformability index (P<0.05,P<0.01.) at 24 h after modelling. As compared with the model rats, ratsin timosaponin BII 40 mg/kg group showed significant increase inerythrocyte deformability index and decrease in erythrocyte aggregationindex (P<0.05, P<0.01),

III. Discussion and Summary

Hemorheology is a science concerning blood fluidity, aggregation,coagulability and blood cell deformability. Hemorheological parameterssuch as whole blood specific viscosity, plasma specific viscosity,erythrocyte aggregation index and fibrinogen may change in patientssuffering from ischemic cerebrovascular disease. Therefore, improvementsin hemorheology, including decrease of blood viscosity and erythrocyteaggregation, and increase of erythrocyte deformability, are crucial inprevention and treatment of ischemic cerebrovascular disease. In ourstudy, rats were subcutaneously injected with a large dose of adrenalineto simulate anger and anxious status and were placed in ice water tosimulate chill status. In this way, an acute blood stagnation model withfeatures of viscous, thick, coagulated and aggregated blood wasreplicated. From the results it is shown that timosaponin BII couldsignificantly inhibit erythrocyte aggregation, increase erythrocytedeformability and reduce whole blood and plasma viscosities at high,middle and low shear rats in acute blood stagnation model rats,suggesting that the drug can significant improve the hemorheology.

Example 3 Effect of Timosaponin BII on Inflammatory Factors in BrainTissues in Cerebral Ischemia-Reperfusion Rats

The embolic thread method was employed in the preparation of cerebralischemia-reperfusion rat model, and ELISA was used to determine thelevels of IL-1β, TNF-α, IL-10 and TGF-β in each group so as toinvestigate the protective effect of timosaponin BII on inflammatoryfactors in cerebral ischemia-reperfusion model rats.

I. Method 1. Grouping and Dosing

The rats were randomly divided into 6 groups based on body weight, i.e.the sham-operation group, model group, nimodipine (12 mg/kg) group,timosaponin Blat 10 mg/kg group and 40 mg/kg group. All drugs wereformulated with 0.5% CMC. Rats of each group were used for experimentsafter 2 days' observation and raising. Continuous administration of thedrugs by gavage was performed for 5 days with a daily volume of 10ml/kg. The sham-operation group and model group were given the samevolume of a 0.5% CMC solution once a day. The operation was carried outone hour after drug administration in the morning of the fifth day.

2. Modelling Method

Middle cerebral artery obstruction (MCAO) model was prepared asdescribed by Koizumi and Nagasawa. Rats were anesthetised byintraperitoneal (ip) injection with 10% chloral hydrate at 0.35 g/kg andwere fixed in supine position. After local sterilization, the operationbegan. The right common carotid artery (CCA), right internal carotidartery (ICA) and external carotid artery (FCA) were separated andthreads were embedded for future use. The ECA and CCA were ligated. Soonafter the distal end of the ICA was closed by an artery clamp, aincision was made at the bifurcation of the ECA and ICA, and one nylonthread with one end heated to become bulb-shaped (0.25 mm in diameter,marked at 2 cm from the bulb end, and the anterior end of the embolicthread was treated with paraffin for future use) was inserted. After thethread was inserted into the ICA, the thread and the ICA inlet wereslightly ligated, and then the artery clamp was released. The nylonthread was inserted further into the ICA and was slightly withdrawn whenthere was a little resistance, until it reached the depth of about18.5±0.5 mm, causing MCA obstruction and hence cerebral-ischemia. Theinlet was ligated again, leaving 1 cm long nylon thread outside. Themuscles and skin were sutured, and the rats were injectedintraperitoneally with gentamicin sulfate at 0.4 ml/per rat. After 3 h,the thread end was gently pulled outwards until there was resistance,leading to MCA reperfusion. Thus, the modelling was completed. In thesham-operation group, only right CCA was ligated, with no incision andthread insertion. The inclusion criteria: 3 h after the ischemia,animals that showed signs of contralateral forelimb twisting, walkingaround in a circular path or falling down in a contralateral directionwhile walking were included. Animals that did not show these signs orwere still unconscious after 3 h were excluded.

3. Preparation of Tissue Homogenates

The animals were decapitated 3 h after ischemia and 21 h afterreperfusion. The olfactory bulb, cerebellum and low brain stem wereremoved and the rest of the right hemisphere was homogenized in normalsaline at 4° C. to a concentration of 10%.

4. ELISA Assays for IL-1β, TNF-α IL-10 and TGF-β

(1) Establishment of Standard Curve

Eight standard wells were established, and each was added 100 ul ofsample dilution solution. To the first well was added 100 ul ofstandard, mixed well, and then 100 ul was pipetted to the second well.This double diluting process was repeated until the seventh well.Finally, 100 ul was pipetted from the seventh well and discarded so thatthe volume in each well was 100 ul. The eighth well served as the blankcontrol.

(2) Loading

150 ul of sample was added to each sample well.

(3) The reaction plate was mixed well and placed at 370 for 120 min.

(4) Plate Washing

The reaction plate was thoroughly washed with washing solution 4-6times, and blotted with filter paper.

(5) To each well was added 50 ul of a first antibody working solution,and the plate was placed at 37° for 60 min.

(6) Plate washing: see above.

(7) To each well was added 100 ul of an enzyme-conjugated antibodyworking solution and the plate was placed at 37° C. for 60 min.

(8) Plate washing: see above.

(9) To each well was added 100 ul of a substrate working solution, andthe plate was placed in dark at 37° for 5-10 min.

(10) To each well was added 1 drop of a stop solution and was mixedwell.

(11) The absorbance at 492 nm was read.

5. Result Calculation

Calculation was done after subtracting the blank value from all ODvalues. The OD values for the standard at 1000, 500, 250, 125, 62, 31,16, and 0 PG/ml were plotted on a semi-log paper, to obtain a standardcurve. The level of the corresponding inflammatory factor could bedetermined from the standard curve based on the OD value of the sample,

6. Statistical Analysis

The experimental data were expressed as is. The results were analyzedusing SPSS software and t-test was used to perform comparison amonggroups.

II. Results Effect of Timosaponin BII on Inflammatory Factors inCerebral Ischemia-Reperfusion Rats

TABLE Effect of Timosaponin BII on levels of IL-1β, TNF-α, IL-10 andTGF-β in ischemia-reperfusion rats ( x ± s) Dosage IL-1β TNFα IL-10TGF-β Group mg/kg n (Pg/ml) (Pg/ml) (Pg/ml) (Pg/ml) Sham-operation — 8 9.42 ± 0.94 7.91 ± 0.36  3.55 ± 3.03 12.89 ± 4.51 Model — 8 10.95 ±0.74^(ΔΔ) 8.74 ± 0.70^(ΔΔ) 16.75 ± 3.45^(ΔΔ) 17.69 ± 2.84^(Δ) Nimodipine12 8  9.01 ± 0.81** 7.92 ± 0.56*  4.98 ± 3.54** 12.01 ± 2.54**Timosaponin BII 10 8  9.70 ± 0.30** 8.44 ± 0.62 14.49 ± 11.78 12.70 ±3.57* Timosaponin BII 40 8  7.98 ± 0.80** 7.65 ± 0.61** 11.19 ± 5.16*10.30 ± 3.79** Notes: ^(Δ)P < 0.05, ^(ΔΔ)P < 0.01 vs. the sham-operationgroup; *P < 0.05, **P < 0.01 vs. the model group

The experimental results indicated that as compared with thesham-operation group, the levels of pro-inflammatory factors IL-1β andTNF-α in brain tissue of cerebral ischemia-reperfusion model ratssignificantly increased, and the levels of protective inflammatoryfactors IL-10 and TGF-β also increased significantly, and thedifferences were statistically significant. Positive control ofNimodipine and timosaponin BII at 40 mg/kg significantly reduced thelevels of these inflammatory factors and thus had obvious protectiveeffects in the inflammation responses in ischemia-reperfusion modelrats.

1. Use of timosaponin BII in the preparation of a medicament or productfor the prevention or treatment of stroke (apoplexy).
 2. Use ofTimosaponin BII according to claim 1, characterized in that the purityof Timosaponin BII is ≧90%.